Bioanalytical methods in doping controls: a review

ABSTRACT

The analytical and technological approaches employed in doping analysis are constantly reviewed and updated to allow for keeping pace with progresses in pharmaceutical and medicinal research and the therein inherent options of misuse as performance enhancing drugs or methods. Enormous changes, improvements, and developments have been achieved in recent years, particularly, but not exclusively, in the bioanalytical sector. Several of these new strategies are examined systematically in this review using examples from the World Anti-Doping Agency (WADA) list of banned substances and methods. The review includes, among others, the application of sophisticated new in-vitro models mimicking multi compartment models, investigation into new long-term metabolites for anabolic agents, the impact of a distinct gene mutation on the analysis of erythropoietin, studies on the development of new therapeutic protein-based drugs with myostatin inhibiting properties, methods applying the new molecular biological section used to uncover gene doping, and finally new approaches uncovering the prohibited use of autologous blood transfusion. All of these challenges and investigations support the ongoing progress in modern doping controls in the future and will help to fill the gap between the advance of cheating athletes and sport drug testing.

1. Introduction

The properties and quality of doping control are a critical and important aspect in maintaining fairness and integrity of sports by ensuring the absence of prohibited substances in an athlete`s specimen. The basic requirements and regulations for drug testing in sport are globally organized by the World Anti-Doping Agency (WADA) and its national counterparts [1,2]. The detection of performance-enhancing drugs (PEDs) requires advanced bioanalytical techniques that are highly sensitive, specific, and reliable on the one hand, and being comprehensive, cost effective, and fast on the other hand. Obviously, the established methods represent a compromise for all of these criteria, with the endless potential to improve the one and/or the other part. Considering the very special requirements in this analytical field with a very high number of inconspicuous (negative) samples and only a low number of suspicious (positive) samples, most of the progress and developments originate from doping control laboratories, which apply the general improvements in analytical chemistry on their dedicated problem. This represents a very dynamic process in which the frame conditions are set by the WADA on the one hand and the technical requirements/limitations on the other hand [2–4]. Noteworthy, any substantial progress also from other related analytical disciplines was adapted in sport drug testing assays as well. In most cases and whenever possible, established assays in doping controls are preferably based on chromatographic separations (gas or liquid chromatography, GC resp. LC) with mass spectrometric (MS) detection, ligand-binding assays (LBA), immunoassays, electrophoretic methods and recently also methods based on molecular biologic principles (e.g., polymerase chain reaction PCR) [4,5]. During the last years, more and more of these methods evolved remarkably and also hybrid assays exists combining the benefits of different orthogonal approaches. Noteworthy, apart from the aforementioned analytical methods also sophisticated bioanalytical techniques e.g., for in-vitro production of metabolites, production of specific antibodies and other approaches were involved in the recent research topics for sports drug testing. This review provides an overview of the key bioanalytical methods used in doping control, highlighting their principles, applications, and advancements.

2. Bioanalytical techniques

The term “bioanalytical methods” is not accurately defined in a completely harmonized manner, thus, various assays and approaches potentially fall under this definition. The present review will mainly focus on the following (bio)-analytical approaches as shown also in Figure 1): Ligand-binding-assays (LBA) including enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA) etc., gel-based methods (SDS-PAGE, SAR-PAGE, Western blot, etc.), hybrid LC-MS (combining bioanalytical pretreatment with LC-MS analysis) and various variations of PCR assays. Furthermore, several new approaches which facilitate effective doping control analysis using bioanalytical methods (e.g., sophisticated new in-vitro models) are also included in this review. The following paragraph will illustrate important progress and new bioanalytical approaches in doping controls approximated with the occurrence in the WADA Prohibited List.

Figure 1.

Selected bioanalytical techniques used in doping controls (a) ligand-binding-assays (LBA, incl. ELISA, RIA), (b) polymerase chain reaction (PCR) methods, (c) gel-based methods (SDS-PAGE, SAR-PAGE, Western blot, etc.) and (d) chromatography coupled to mass spectrometry (GC/LC-MS).

3. Non-approved substances

In this section, recent developments concerning substances which currently do not hold a valid approval and for which no other appropriate classification in WADA’s Prohibited List exists are summarized. The recent implementation of high-resolution mass spectrometric detection assays with untargeted analysis design enables data acquisition with the option to retrospectively evaluate the dataset for previously unknown compounds from this section [3,6–11]. The evaluation of already acquired data for the presence of new drugs facilitates the collection of information on the relevancy and hazard of previously unknown (and presumably performance-enhancing) substances. Although these approaches are also limited (due to unknown metabolic fate or suboptimal analytical conditions, etc.), the herewith obtained information may serve as a so-called “digital matrix” for further investigation if the question arises regarding new, non-approved drugs [5].

Another important technical progress represents the in-vitro production of relevant metabolites by sophisticated new models [12–18]. These models are able to simulate several different compartments of the human organism by combining the respective tissue cells (separated in compartments) in one experiment and model the metabolism in the human body in much more realistic conditions compared to earlier models [19]. This has been used already successfully in doping controls with non-approved substances, aiming at reducing the need for in vivo studies with these compounds. Exemplarily, the metabolism of the non-approved selective androgen receptor agonist RAD-140 is named here [20]. Such three-dimensional multicompartment models can consist of cells from skin, liver, kidney, lung, etc., and enable the ongoing incubation over several days (weeks) under consideration of all chosen types of cells [20]. In these “organ-on-a-chip” experiments, the conditions in the human body are simulated very effective and enable the in-vitro simulation of complex metabolic processes generating metabolites which were not available by former classical in-vitro models [21]. This is of utmost importance in doping controls due to the demand for adequate reference material in case of confirmation analysis.

4. Anabolic agents

The high number of adverse analytical findings that fall under the group of anabolic agents each year alone shows the high relevance of this substance class in sport. In addition to the classic steroidal substances, which can be classified structurally as derivatives of testosterone, more and more non-steroidal substances are also listed here [1]. Many studies in recent years have been concerned with elucidating the metabolism of steroids in such a way that metabolites can be found that can be detected particularly long after application [15,17,18,22–28]. This enables the detection of abuse over a long period of time and increases the efficiency of doping control enormously. Such long-term metabolites have led, among other things, to a very large number of positive results being found, particularly in the reanalysis of Olympic samples in recent years. In detail, new metabolites for dehydrochloromethyltestosterone, stanozolol and oxandrolone yielded in sum more than 110 (!) findings only in the reanalysis of samples from London and Beijing (2012 and 2008) [29]. Noteworthy, the analysis and metabolism of classical steroidal anabolic agents are still subject of many and extensive investigations [21,30–39]. Especially, the shift from urine to blood (serum) specimens for the evaluation of steroid profiles represents an important trend during the last years [40–53]. This represents a remarkable recent development due to the very low concentrations of steroids in blood compared to urine samples. This complicates the analysis of blood samples considerably and is enabled due to the increased sensitivity of modern mass spectrometer only. Generally, the utilization of doping control blood samples (more expensive than urine) for more than one type of analysis (growth hormone, EPO, etc.) is desirable and may justify the increasing number of this type of samples. While urinary steroid profile analysis is mainly performed with GC-MS/MS after derivatization, the blood samples were often analyzed by LC-MS/MS without derivatization [45,50,51]. In this regard, Langer et al. show a remarkable comparison of different approaches for the detection of testosterone undecanoate misuse including their method inherent benefits and shortcomings [44].

5. Peptides and proteins

Regarding peptides and proteins, bioanalytical approaches are extensively established due to the fact that these target analytes are mainly categorized as “biomolecules.” Thus, most of the methods for processing, extraction, purification, and detection apply bioanalytical procedures [3]. While small molecules (such as steroids, stimulants, etc.) are often treated according to their physico-chemical properties, such as polarity, acidity or basicity, macromolecules (peptides, proteins etc.) own additionally the potential to use biological properties which enable effective processing. Here, mainly the ability of biomolecules to reversibly bind to ligands is utilized to isolate or separate target substances (or classes of substances) from the matrix. Ligand binding is realized most often by immunogenicity (extraction with antibodies) or receptor binding, with the antibody or receptor immobilized previously. In the following several new approaches will be listed and explained, which are mainly based on ligand binding.

Since the last decades the misuse of erythropoiesis stimulating agents (ESA) yields a constant number of adverse analytical findings, which is most likely due to effective performance enhancing properties of ESAs in endurance sport. The ESAs are usually detected by means of SAR-PAGE analysis and include all available ESA analogs in the assay. Recently, in the analysis of recombinant erythropoietin beta, it has been noticed that there are certain genetic predispositions that require consideration in the interpretation of SAR-PAGE results [54]. A mutated EPO gene was identified, which has a 27 amino acid longer sequence and leads to the characteristic double band on the gel. Since then, this has been referred to as a c.577del EPO variant (rs369859204), which has a frequency of less than 0.5% in the examined Chinese population pool [54], and confirmation whether an athlete is a carrier of the c577del variant is necessary whenever a suspicious test result for recombinant human EPO is produced [54–57]. Recently, also the additional control with an internal standard on the gel enables sophisticated EPO analysis with consideration of the c.577del variant [58]. Here the additional usage of an Anti-VAR-EPO antibody as a ”reverse immunopurification” approach enables the reliable consideration of the variant c.577del gene for the confirmatory analysis of recombinant EPO. Another interesting new approach to face the detection of the gene variant c.577del directly from urine samples or alternatively dried blood spots is presented recently by Leuenberger et al. Here it is was shown that the Sanger sequencing of the EPO gene was possible in the majority of the urine samples (85%) and all of the dried blood spot samples [59]. An additional blood sampling from the athlete is not required accordingly.

Another important aspect to manipulate muscle growth that has recently emerged is influencing (inhibiting) the myostatin or activin-A axis in the human body [60]. By inhibiting the synthesis of negative growth factors for muscles, the muscle growth of athletes can be potentially increased. This strategy is called: “inhibiting the inhibitors,” and leads to increased muscle cell protein synthesis, decreased degradation, enhanced mitochondrial biogenesis, and preserving muscle function [60]. In particular, myostatin antagonists (antibodies, ligands, etc.) at the protein level should be mentioned here. These clearly exogenous substances (e.g., fusion proteins) can be bioanalytically detected by various methods, whereby liquid chromatography coupled to mass spectrometry, ligand-binding assays or gel-based methods (or combination of all) have proven to be advantageous [61–67]. The liquid chromatography/mass spectrometry-based methods use immuno-purification by means of magnetic beads (loaded with receptors-or antibodies against the target protein), enzymatic hydrolysis (bottom-up) and analysis of the diagnostic peptides with high resolution mass spectrometry [65,67]. In the gel-based protocols also the immune-precipitation enables the clean-up prior to the SDS-gel separation and detection with western blotting [66,68]. Both principle approaches enable the sensitive detection of the respective prohibited proteins in doping control specimens (mostly serum or plasma) in ng/ml range. Noteworthy, these therapeutic peptides or proteins (e.g., Domagrozumab, Bimagrumab, Landogrozumab, Ramatercept (ACE-031), MYO-029 (Stamulumab), Follistatin, ACE-083, SRK-015 (Apitegromab), Trevogrumab, Garetosmab, Sotatercept, Luspatercept, GYM329, Apitegromab, Taldefgrobep, Elritercept) have not reached the status of approved drugs in most cases and most of them might never reach. Nevertheless, misuse in professional as well as nonprofessional sports is not excluded or unusual and the black market shows the availability [68]. Table 1 shows a number of drug candidates in this context including their mechanism of action and development status.

Table 1.

Overview of protein-based myostatin inhibitors.

Drug candidates	Brand name	Drug class	Molecular target(s)	Manufacturer	Therapeutic application(s)	Performance enhancing effect(s)	Clinical development	Clinical approval
Stamulumab (MYO-029)	–	Therapeutic antibody	Myostatin	Wyeth Pharmaceuticals	Muscular Dystrophy	↑ Muscle mass	Phase 2	Discontinued
Domagrozumab (PF-06252616)	–	Therapeutic antibody	Myostatin	Pfizer	Duchenne Muscular Dystrophy	↑ Muscle mass	Phase 2	Discontinued
Landogrozumab (LY2495655)	–	Therapeutic antibody	Myostatin	Eli Lilly	Muscle wasting disorders	↑ Muscle mass	Phase 2	Discontinued
Trevogrumab (REGN1033, SAR391786)	–	Therapeutic antibody	Myostatin	Regeneron Pharmaceuticals	Muscle atrophy/Obesity	↑ Muscle mass	Phase 2	–
Bimagrumab (BYM-338)	–	Therapeutic antibody	ActRIIA/B	Novartis/Versanis Bio/Eli Lilly	Cachexia in different muscoskeletal diseases	↑ Muscle mass	Phase 3/Phase 2	–
GYM329 (RG6237, RO7204239)	–	Therapeutic antibody	Latent Myostatin	Chugai Pharmaceutical/Roche	Spinal Muscular Atrophy/Obesity	↑ Muscle mass	Phase 2/3	–
Apitegromab (SRK-015)	–	Therapeutic antibody	Latent Myostatin	ScholarRock	Spinal muscular atrophy	↑ Muscle mass	Phase 3/Phase 2	Expected 2025
Ramatercept (ACE-031)	–	ActRIIB-Fc fusion protein	Myostatin and other ActRIIB ligands	Acceleron Pharma	Duchenne muscular dystrophy	↑ Muscle mass	Phase 2	Discontinued
ACE-2494	–	ActRIIB-Fc fusion protein (modified)	Myostatin and other ActRIIB ligands	Acceleron Pharma	Neuromuscular disorders	↑ Muscle mass	Phase 1	Discontinued
Taldefgrobep alfa (BHV-2000, Talditercept alfa, RG-6206)	–	Adnectin	Myostatin	Bristol-Myers Squibb/Hoffmann-La Roche/Biohaven	Spinal muscular atrophy/Obesity	↑ Muscle mass	Phase 3/Phase 1	–
Garetosmab (REGN2477)	–	Anti-activin A antibodies	Activin A	Regeneron	Fibrodysplasia ossificans progressiva		Phase 3	–
Sotatercept (ACE-011)	Winrevair	ActRIIA-Fc fusion protein	Activin A	Acceleron Pharma/Celgene; Merck Sharp & Dohme	Pulmonary arterial hypertension	↑ Erythropoiesis	Completed	2024
Elritercept (KER-050)	–	ActRIIA-Fc fusion protein	Activin A	Keros Therapeutics	Anemia	↑ Erythropoiesis	Phase 2	–
Luspatercept (ACE-536)	Reblozyl	ActRIIB-Fc fusion protein (modified)	GDF-11	Acceleron Pharma/Celgene; Bristol Myers Squibb	Anemia	↑ Erythropoiesis	Completed	2019/2020

*References: Domagrozumab [67,69–77], Bimagrumab [78,79]Landogrozumab [80], Ramatercept (ACE-031) [81,82], MYO-029 (Stamulumab) [83–85], Follistatin [65,68], ACE-083 [86–89], SRK-015 (Apitegromab) [90–92], Trevogrumab [93], Garetosmab [93], ACE-2494 [94], Sotatercept [95], Luspatercept [96] GYM329 [80], Apitegromab [97], Taldefgrobep [98], Elritercept [99].

Another focus in the analysis of doping-relevant peptides/proteins is the progress in the determination of parameters in the GHRH/GH/IGF-I axis. The abuse of growth hormone is prohibited in sport at all times, but the detection of recombinant GH is complicated by the fact that it is also an endogenously produced hormone, which necessitates differentiating exogenous and administered GH from naturally produced isoform compositions [100]. A suitable test has been established more than ten years ago [100], and further studies have also shown that other complementary biomarkers exit that allow for extending the detection window for GH administrations. These biomarkers are IGF-I and P-III-NP and their joint determination with subsequent monitoring in a dedicated module of the athlete biological passport shows promising results [101–104]. In the beginning, ligand-binding assays, which can be well standardized in the laboratories, were generally used for the precise and reliable quantification required for this. However, because the determination by LC-MS is considered to be more advantageous and superior overall, there have recently been several studies on the determination of IGF-I and also P-III-NP from blood samples using LC-MS [105–110]. Crucial requirement for obtaining comparable results within the participating laboratories is the availability and distribution of reliable reference material. Especially the usage of stable isotope-labeled material used as internal standards improves the data considerably [106].

6. Metabolic modulators

Section 4 (Metabolic modulators) of WADA Prohibited List contains a number of very diverse substances that differ greatly in terms of their chemical structure and biological effect in the body. The analysis of these substances is mainly realized by classical chromatographic separation coupled to mass spectrometry for many years already. Noteworthy, the metabolism of these substances has been the subject of many studies in recent years [111,112]. For example, the metabolism of clomifene, which is banned as a selective estrogen receptor modulator (SERM) in sport at all times, has also been investigated. This drug is used therapeutically to treat infertility in women, but it is also known to lead to better laying characteristics in chickens. It has been shown that it is possible to be contaminated (and thus tested positive) with clomifene by eating eggs if the hens have been treated beforehand. However, the metabolite pattern in the urine of human volunteers allows a differentiation from patients who were directly treated with clomifene. Noteworthy, these very complex studies can ultimately work to an athlete’s advantage and sophisticated data interpretation is enabled [113,114].

7. Narcotics

While narcotics as morphine and its derivatives belong to the first-generation doping substances used and analyzed already in the beginning of doping controls in the 1960s, the number of positive findings with narcotics is decreasing over the time. While the majority of narcotics belong to the small molecules which are usually not in the scope of bioanalytical methods, new bioanalytical developments in this field are rare. The detection of this prohibited drugs is well investigated and the existing methods are efficient and sensitive. But recently, the analysis of the peptide-based natural dermorphine and its synthetic analogs has been considered in doping controls in humans (as well as in race horses) due to the implementation into the WADA Monitoring Program [115]. Thus, several analytical assays as well as metabolism studies were published for dermorphine and its analogs [116–125]. The dermorphine peptides own strong narcotic properties, without showing the severe side effects (tolerance, addiction, etc.), thus the misuse in sports might be relevant. Several different endogenous and synthetic dermorphin analogs are known and test methods were published recently [123,126]. The amino acid sequences and their respective origin (endogenous and/or synthetic) are shown in Table 2. Considering the obvious complexity and similarity of the different types of analogs which are potentially of endogenous origin, a reliable and meaningful analysis is not straight forward and requires sensitive and specific methods for each target peptide. In addition, the metabolic fate and the interindividual relation of these peptides is largely not known so far. The included amidation of the C-terminus and the incorporation of D-amino acids and/or further unnatural amino acids will induce a prolonged half-life in the circulation due to increased resistance against endogenous proteases.

Table 2.

Dermorphin peptides.

Dermorphin analog	Amino acid sequence	Origin
[D-Ala2]Dermorphin	Tyr-dAla-Phe-Gly-Tyr-Pro-Ser-NH2	endogenous
[L-Ala2]Dermorphin	Tyr-Ala-Phe-Gly-Tyr-Pro-Ser-NH2	endogenous
[D-Ala2, Hyp6]Dermorphin	Tyr-dAla-Phe-Gly-Tyr-Hyp-Ser-NH2	endogenous
[D-Ala2, Lys7]Dermorphin	Tyr-dAla-Phe-Gly-Tyr-Pro-Lys-NH2	endogenous
[D-Ala2, Trp4, Asn7]Dermorphin	Tyr-dAla-Phe-Trp-Tyr-Pro-Asn-NH2	endogenous
[D-Ala2, Trp4, Asn7]Dermorphin-OH	Tyr-dAla-Phe-Trp-Tyr-Pro-Asn	endogenous
[D-Ala2, Trp4, Asn7]Dermorphin-1-5OH	Tyr-dAla-Phe-Trp-Asn	endogenous
[D-Arg2, Sar4]Dermorphin	Tyr-dArg-Phe-Sar-Tyr-Pro-Ser-NH2	synthetic
[D-Arg2, Sar4]Dermorphin-1–4	Tyr-dArg-Phe-Sar	synthetic
[L-Arg2]Dermorphin	Tyr-Arg-Phe-Gly-Tyr-Pro-Ser-NH2	synthetic
[D-Arg2]Dermorphin	Tyr-dArg-Phe-Gly-Tyr-Pro-Ser-NH2	synthetic
[D-Arg2]Dermorphin-1–4	Tyr-dArg-Phe-Gly-NH2	synthetic
[D-Arg2]Dermorphin-1–4-OH	Tyr-dArg-Phe-Gly	synthetic
[D-Arg2]Dermorphin-1–5	Tyr-dArg-Phe-Gly-Lys-NH2	synthetic
[D-Arg2, βAla4]Dermorphin-1–4	Tyr-dArg-Phe-βAla-NH2	synthetic
[D-Arg2, βAla4]Dermorphin-1-4OH	Tyr-dArg-Phe-βAla	synthetic
[Dmt1, D-Arg2, Lys4]Dermorphin	(CH3)2Tyr-dArg-Phe-Lys-NH2	synthetic

Hyp: Hydroxyprolin, Sar: Sarkosin, Dmt: Dimethyltyrosin.

8. Gene doping

Gene doping is a prohibited method according to the actual regulations of WADA and it refers mostly to the misuse of gene therapy techniques to enhance athletic performance by artificially altering genes or gene expression [1]. Unlike traditional doping methods and substances, which involve the use of performance-enhancing drugs such as recombinant peptides or antibodies, gene doping targets the athlete’s genetic makeup, making it much harder to detect. Existing and established (classical) analytical methods largely fail to uncover the manipulation on genetic level. Thus, the detection of gene doping requires sophisticated assays that can identify unnatural modifications (such as transgenes or chemically-modified nucleotides) in an athlete’s genome or transcriptome accordingly [127–134]. Likely candidates for illicit transgenic expression are strength and/or endurance enhancing factors such as EPO, FST, IGF-I, VEGF and GH and genomic manipulations (e.g., via CRISPR/Cas approaches) could target, e.g., the MSTN coding sequence (for a knock-out) or regulatory sequence elements of the previously mentioned genes. For the detection of artificial genes, most assays rely on quantitative real-time PCR on unnatural, intron-lacking exon-exon junctions of the respective transgene [127–134]. While urine is the mainly used matrix in most doping control assays, these urinary specimens represent a more unusual matrix in classical assays for molecular biology, thus, the sample extraction protocols have to be adapted to enable the reliable purification of low amounts of DNA from urine [4]. Noteworthy, the analysis of whole blood samples is recommended also due to the potential enrichment of transgenes from white blood cells [135]. This is especially true under consideration, that the available volume of urine is limited due to various different assays required for complete doping analysis. Both targeted and un-biased next-generation sequencing (NGS) approaches can enable the detection of several different gene doping targets simultaneously and could be powerful screening methods for the detection of even unknown exogenous transgenes or genomic manipulations [129,133,136,137]. Such a multiplexed method was also applied for the analysis of two black products available via internet which were found to contain alleged plasmid products with transgenic EPO. Up to our knowledge, this is the first confirmed finding of a commercially available gene doping product and verifies the relevancy of developing effective methods for uncovering the misuse [138].

After the discovery of the new fascinating genetic tool to manipulate the expression of proteins and peptides by means of clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas)-based interaction, also its potential for manipulation of athletic performance was realized as serious hazard in professional sport [139–144]. Several assays were developed with focus on this gene engineering tool and its various applications in sports drug testing [139–141,143]. While some assays are designed to uncover the direct misuse of CRIPSR/Cas as doping tool [140,143,145], others use the accurate cleavage function of the system to uncover the application of other transgenes to the organism [139,141].

9. Blood doping

Blood doping involves increasing the number of red blood cells (RBCs) to enhance athletic performance by improving oxygen delivery to muscles. The artificial increase of the total hemoglobin amount by means of autologous blood transfusion (prohibited at all times) represents still a considerable challenge for all doping control laboratories. The uncovering/detection of such autologous transfusions represents one of the major challenges in the investigations of new bioanalytical strategies. Especially, the fact that the transfused blood belongs to the same individuum and is diluted with the actually circulating blood in the organism hinders the simple detection. Several completely different approaches have been developed to uncover transfusion of the previously drawn blood prior to the competition [146–154]. While some studies apply metabolomic approaches [146,147], others use changes in the microRNA pattern [151–153,155] or changes in the blood cells surface [148,154]. The methods using RNA biomarkers were shown to even detect micro-doses of erythropoietin by considerable changes in the expression of the enzymes 5’-aminolevulinate synthase 2 (increase up to 300% after EPO treatment) and carbonic anhydrase 1 (increase up to 200% after EPO treatment) [156]. Additionally, in horse doping the monitoring of substantial biomarkers were found to be diagnostic for the indirect detection of EPO analogs in horses using the expression of the aforementioned enzymes [157]. All of them show promising preliminary results and will potentially help to support the data collected in the already established athletic biological passport (ABP) [158]. The ABP is a long-term monitoring tool that tracks an athlete’s hematological parameters over time. By establishing an individual baseline, the ABP can detect abnormal fluctuations in blood markers, such as hemoglobin concentration and reticulocyte (immature RBCs) count, indicative of blood doping. The ABP is highly effective in identifying blood manipulations, even without direct evidence of doping substances [158]. Another way to increase the number of RBCs is homologous blood transfusion with blood from a compatible donor. The detection here is much easier, because the blood samples usually contain two different RBC populations. While earlier assays use flow cytometry for detection, recently also SNP-based genotypic strategies were applied here [159–161]. Noteworthy, also the ABP is able to indirectly uncover these methods.

10. Conclusion

As described in detail in the previous chapter, there is an enormous number of new bioanalytical developments, particularly in the field of modern doping analysis within the last years. This is particularly worth mentioning because this progress usually has to be achieved exclusively by the laboratories carrying out these analyses themselves. In principle, there is no direct or further commercial benefit from this further development, as these analyses are generally carried out here exclusively. Nevertheless, research in this area is extremely important in order to do justice to the increasingly complex possibilities and pitfalls of athletes in an unregulated environment [162,163]. However, in order to keep pace with these challenges, the laboratories are also dependent (in addition to personnel and financial support) on interdisciplinary cooperation with other scientific and industrial disciplines, which will play an equally important role in the ongoing progress and successful outcome of the research.

11. Future perspective

The future challenges in the contemporary analysis of doping samples are (as the past has shown) strongly dependent on developments in pharmaceutical drug research. Newly developed drugs that have the potential to improve performance necessitate consideration by anti-doping organizations and anti-doping laboratories are urged to establish adequate analytical approaches accordingly. Such new analyses can be implemented very quickly in individual cases if the substances can be easily detected using established methods (e.g., new stimulants, anabolic steroids etc.). In these cases, all that is needed is the availability of the reference substances and information on the metabolism or the urinary target analyte. However, it has also been shown that the introduction of completely new forms of medication (which are sensible and effective from a medical point of view) also poses major challenges for laboratories. Many of the bioanalytical procedures required for this are not established here as standard, neither in terms of instruments nor personnel. Prompt implementation is therefore much more difficult and involves considerable effort. Examples from the recent past include methods for analyzing protein-based doping substances (e.g., growth hormones, erythropoietin, fusion proteins, etc.). Such analyses are generally much more cost- and time-intensive than the analysis of classic doping substances. The spread of genetic manipulation (so called gene doping) is currently seen as a major threat to sport. Such genetic changes cannot usually be detected using conventional (including bioanalytical) methods because the manipulated exogenous genes cause human-identical protein expressions and, thus, existing methods are not effective. Detection solutions for this have already been developed, but the molecular biological methods used here represent a new technology for laboratories that is not easy to establish.

Funding Statement

This paper was not funded.

Article highlights

• Introduction:

 The global requirements and frame conditions of doping control are regulated by the World Anti-Doping Agency. All bioanalytical developments will finally be approved by national and international organizations.

• Bioanalytical techniques:

 While most of the recent progress has been made by investigation in new detection strategies, also new bioanalytical approaches (e.g., in-vitro models) are mentioned here.

• Non-approved substances:

 The section of substances without any medical approval represents an important topic for research. New prohibited target analytes with performance enhancing properties appear here first.

• Anabolic agents:

 During the last decade anabolic agents produce the highest number of positive findings every year. This is why any progress toward better detection is enormously effective in the fight against doping. This chapter shows the remarkably high number of positive retests of Olympic samples.

• Peptides and Proteins:

 The EPO c.577del variant has significantly changed the interpretation of EPO analysis and the reverse immunopurification method shown here is a very valuable tool for easy determination. Furthermore, there is great potential in the development of antibody-based drugs. Here, analytics must be able to keep pace in a timely manner.

• Metabolic modulators and narcotics:

 Especially dermorphine derivates have shown increasing attention and here several different analogs are described.

• Gene Doping:

 This chapter shows the impressive potential of PCR-based methods to determine gene doping by athletes. While some approaches are still in developing state, other approaches are already effective and in use.

• Blood Doping:

 Although this method is known for several decades already, the final solution to solve this problem is not found yet. Noteworthy, this chapter shows several very promising new approaches (namely RNA biomarker methods in combination with the athlete biological passport) toward the potential detectability in the future.

Disclosure statement

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Writing disclosure

No writing assistance was utilized in the production of this manuscript.